A multidisciplinary study on the effects of phloem-limited viruses on the agronomical performance and berry quality of Vitis vinifera cv. Nebbiolo

A multidisciplinary study on the effects of phloem-limited viruses on the agronomical performance and berry quality of Vitis vinifera cv. Nebbiolo
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  A multidisciplinary study on the effects of phloem-limited viruses on the agronomical performance and berry quality of  Vitis vinifera  cv. Nebbiolo Marzia Giribaldi a , Micol Purrotti a , Davide Pacifico b , Deborah Santini b , Franco Mannini b ,Piero Caciagli b , Luca Rolle c  , Laura Cavallarin a , Maria Gabriella Giuffrida a, ⁎  , Cristina Marzachì b a Istituto di Scienze delle Produzioni Alimentari, National Research Council, Grugliasco (TO), Italy b Istituto di Virologia Vegetale, National Research Council, Torino, Italy c Di.Va.P.R.A.-Microbiologia Agraria e Tecnologie Alimentari, Grugliasco (TO), Italy A R T I C L E I N F O A B S T R A C T Available online 11 August 2011 Viral infections areknowntohavea detrimental effect ongrapevine yield and performance,but there is still a lack of knowledge about their effect on the quality and safety of endproducts.Vines of   Vitis vinifera  cv. Nebbiolo clone 308, affected simultaneously by Grapevine leafroll-associatedvirus1(GLRaV-1),GrapevinevirusA(GVA),and Rupestrisstempitting associatedvirus (RSPaV), were subjected to integrated analyses of agronomical performance, grapeberry characteristics, instrumental texture profile, and proteome profiling.The comparison of performance and grape quality of healthy and infected vines cultivatedin a commercial vineyard revealed similar shoot fertility, number of clusters, total yield,with significant differences in titratable acidity, and resveratrol content. Also some textureparameters such as cohesiveness and resilience were altered in berries of infected plants.The proteomic analysis of skin and pulp visualized about 400 spots. The ANOVA analysis on2D gels revealed significant differences among healthy and virus-infected grape berries for 12 pulp spots and 7 skin spots. Virus infection mainly influenced proteins involved in theresponse to oxidative stress in the berry skin, and proteins involved in cell structuremetabolism in the pulp.© 2011 Elsevier B.V. All rights reserved. Keywords: 2D electrophoresisGrape qualityInstrumental texture analysisMass spectrometryViral infection 1. Introduction Due to its economic importance, the wine-making industryoccupies a leading position in Italian agriculture. Some of theworld'smostappreciatedandvaluedredwines,suchasBaroloand Barbaresco, are produced in Piedmont from Nebbiolograpes. Grapevines in this region are threatened by severalviruses and virus-like pathogens [1]. Among these,  Grapevineleafroll associated virus-1  (GLRaV-1; family  Closteroviridae , genusAmpelovirus) and Grapevine virus A (GVA;family  Betaflexiviridae ,genus  Vitivirus ), are the causal agents of two importantdiseases,leafroll(LR)andrugose wood(RW)diseases,respec-tively.  Rupestris stem pitting associated virus  (RSPaV; family Betaflexiviridae , genus  Foveavirus ), also present innorth-westernItalian vineyards, induces few, if any, symptoms in singly-infected  Vitis  spp., but, as it replicates in the plant, it cancontribute to the development of RW in the presence of other viruses, such as GVA [2].  J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 1 ) 3 0 6  –  3 1 5 ⁎  Corresponding author at: CNRIstitutodiScienzedelleProduzioniAlimentari,UOSTorino,ViaRibes,5 – 10010CollerettoGiacosa(To),Italy.Tel.: +39 0125 564035; fax: +39 0125 564942.E-mail address: (M.G. Giuffrida).1874-3919/$  –  see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jprot.2011.08.006  Available online at  Since no natural resistance to viruses has been identified in V.vinifera ,themanagementofviraldiseasesreliesonpreventivecultural practices and the mandatory use of certified virus-freepropagation material [3]. Programs to eradicate the infections are ongoing; meanwhile, however, berries from infected plantsare routinely used for winemaking.The effects of viral infections on grapevine agronomic andoenological performance are still under investigation, sincethe plants' response to infection may be strongly influencedby the virus strain, the plant genotype, and the environment.Leafroll-infected grapevines show a reduction in the overallrate of photosynthesis [4], and a marked reduction in yield in susceptible cultivars [5]. Moreover, a significant decrease of  theoverallqualityofbothgrapesandmustsisreportedforLR-and RW-infected vines [6 – 10].A detailed knowledge of the molecular effects of viralinfection in grapevines is still lacking, despite the recentcompletionof  Vitis genomesequencingprogrammes[11,12].Ingeneral, evolutionary studies suggest that viruses manipulatehost cellular processes for their life cycle [13,14]. In the grapevine, GLRaV-3 infectioninducesanincreasedexpressionof several genes involved in a wide spectrum of biologicalfunctions, including cell defence [15]. In compatible virus- infected grapevines, an increase in the expression of senes-cence-associated genes, and the accumulation of some pro-teinswithknownallergenicproperties[16]areknowntooccur [17].During the last decade, the number of proteomic studiesdevoted to the investigation of grape physiology and winecharacteristics has boosted. The main issues investigated todate are grape berry ripening, grapevine response to bioticstresses,mainlydroughtandsalinity,andtheformationofhazein white wine following thermal abuses. Comprehensive re-views on these issues have been published in 2002 [18,19], 2007[20], 2010 [21]. Despite its potential, proteomics has rarely been applied to study plant – virus interactions [22,23], and the questions surrounding fruits have not been addressed throughthis method.This study reports the first investigation on agronomicperformance, fruit texture and composition, and proteomicchanges occurring in berries of virus-infected Nebbiolo grapesgrown in field conditions. 2. Materials and methods 2.1. Vineyard The experimental vineyard was located in the Langhe area(Neive,Cuneo,Italy),ahillyviticulturalareacharacterizedbyaloamy-clay soil. The vineyard, north-west oriented, wasplanted in 1992 with infected Nebbiolo vines, clone 308,derived from one srcinally infected mother plant carrying GLRaV-1, GVA and RSPaV in mixed infection. In the samevineyard, the healthy progeny of clone 308, free of viruses byheat treatment [6], was planted in the adjacent row (2.5 mdistance). All vines were grafted onto healthy 420 A rootstock,Guyot pruned and vertically trained. Plant density was about5000 vines/ha. The vineyard was subjected to ordinary agro-nomical and disease-control practices. 2.2. Plant selection Inwinter2007,dormantcaneswerecollectedfrom30plantsof both the Nebbiolo clone 308 infected progeny and the healthyprogeny. Total RNA was extracted from 0.1 g of corticalscraping using the Concert ™  Plant RNA Isolation Reagent(Invitrogen) following the manufacturer's instructions. Thephytosanitary status of each plant was checked by Reversetranscription (RT)-PCR of several grapevine-infecting viruses(ArMV, GFLV, GFkV, GLRaV-1, GLRaV-2, GLRaV-3, GVA, GVBand RSPaV) according to Gambino et al. [24]. Twenty healthy andtwenty infected plants,carrying GLRaV-1,GVA andRSPaVinmixedinfection,wereselectedandlabelledforobservationsthroughout the 2008 growing season. 2.3. Sampling Observations were carried out throughout the 2008 growing season on the 20 healthy and 20 infected plants chosen. 2.3.1. Virus detection and quantification InJuly2008,threebasalandthreefullyexpandedapicalleaveswere harvested from the first three shoots of infected andhealthyplants.Atharvest,leafsamplesforviralquantificationwere collected only from infected vines. 2.3.2. Berry chemical composition, physical characteristicsand proteomics At harvest, berries were sampled from the chosen vinesaccording to the different targets of the experiment. Theberries were randomly picked with attached pedicels fromboth sides of each cluster to avoid the effect of shadowing.Berry juice chemical composition was measured on 30 berrysamples for each individual vine. Three groups of threeadjacent plants among those chosen for each sanitary statuswere selected for berry phenol composition (500 berry pools)and instrumental texture analysis (500 berry pools). For eachinstrumental texture test, 30 berries were randomly selectedfrom the 500 berry pools, after visual confirmation that theywere intact. For proteomic analysis, 400 berries (20 berriesfrom each individual plant) were immediately frozen in thevineyard using dry ice, after washing with tap water, andstored at  − 80 °C until analysis. 2.4. Virus diagnosis and quantification Total RNA was extracted from 0.1 g of midribs using theConcert ™  Plant RNA Isolation Reagent (Invitrogen) following the manufacturer's instructions. The health status of eachselected plant sampled in July was checked by Reverse Tran-scription (RT)-PCR, with primers specific for several grapevine-infecting viruses (ArMV, GFLV, GFkV, GLRaV-1, GLRaV-2,GLRaV-3, GVA, GVB and RSPaV), as indicated in [24]. GLRaV-1andGVA titresinthe infectedplants weremeasuredbyreversetranscription TaqMan Real Time PCR (qRT-PCR) on the RNA-dependent RNA polymerase gene of each virus, and expressedas viral genome units per   V. vinifera  glyceraldehyde-3P-dehydrogenase (GAPDH) transcript copy, following indicationsgivenin[25].ForthequantitationoftheGVAisolateinfectingthe Nebbiolo 308 clone, specific primers and a specific probe were 307  J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 1 ) 3 0 6  –  3 1 5  designed, namely GVAPrep347F (5 ′ -CCTACACTCAGCCCGCAAA-3 ′ ), GVAPrep385R (5 ′ -GCGAGTCCTCGGTTTTCGA-3 ′ ),and the probe GVAPrep367P (5 ′ -CCTTGGCTGCTGAGAT-3 ′ ). 2.5. Agronomical performances and berrychemical composition The agronomical performances (bud burst index, shootfertility, yield, cluster number and weight, and berry weight)of healthy and infected vines were assessed individually onthe20selectedvines throughoutthegrowthseason.Budburstindex was analyzed in spring as indicated in [26]. Bud burstindex indicates the degree of bud development after winter dormancy on a scale from 1 to 5, the shoot fertility is thenumber of inflorescences on each shoot at spring, yield is theweight of grapes per vine at harvest.Soluble solids were measured by a portable refractometer,titratableacidityandpHweremeasuredusingtheInternationalOrganization of Vine and Wine methods [27].Phenolic compounds in berry skin extracts were evaluatedusing an UV-1601PC spectrophotometer (Shimazdu ScientificInstruments) as proposed in [28]. Total anthocyanins were expressed as malvidin-3-glucoside chloride and total flavo-noids were expressed as (+)-catechin. Resveratrol was deter-mined by HPLC applying the chromatographic conditionproposed in [29]. Total anthocyanidins, flavonoids and res- veratrol were quantified using external standards supplied byExtrasynthèse (Genay, France).Briefly, the berry skins were manually removed and driedwith adsorbent paper. Afterwards, they were quickly im-mersed in 25 mL of a pH 3.2 buffer solution containing 12%ethanol and 600 mg/L sodium metabisulphite. After homoge-nization at 6000 × g  for 1 min with an Ultraturrax T25 high-speed homogenizer (IKA Labortechnik), the extracts werecentrifuged at 3000 × g  for 10 min at 20 °C. The supernatantswerethenusedforthe determinationof phenoliccompounds. 2.6. Instrumental texture analysis A Universal Testing Machine TAxT2i Texture Analyzer (StableMicro System, Godalming, UK) equipped with a HDP/90platform and a 5 kg load cell was used for mechanical testing of skin and whole berries. Data were acquired at 400 Hz andevaluated using the Texture Expert Exceed software version2.54.The skin hardness, evaluated as resistance to probe pene-tration, was assessed by a puncture test using an SMS P2/Nneedle probe as described in [30]. The skin hardness was expressed in terms of skin break force (N), skin break energy(mJ) and resistance to axial deformation (Young's modulus,N mm − 1 ). The skin thickness ( μ m) was assessed using a P2 flatprobe and a speed test of 0.2 mms − 1 , as in [31], on a skinfragment (ca. 0.25 cm 2 ) manually excised from the side of eachberry.For the Texture Profile Analysis (TPA) test, each wholeberry was compressed in the equatorial position with an SMSP/35 flat probe under 25% deformation, with a waiting timebetweenthe two bites of 2 s, using 1 mm s − 1 as speed test [30].Typical texture parameters,  i.e.  hardness (N), cohesiveness(adimensional), gumminess (N), springiness (mm), chewiness(mJ) and resilience (adimensional) were provided by thesoftware as described in [31]. 2.7. Protein extraction and 2D electrophoresis 100 berries (5 berries per plant) were cut, deseeded and peeledwhile frozen, the mesocarp (pulp) and pericarp (skin) wereseparatedandfinelygroundinamortarwithapestle,working in liquid nitrogen to ensure sample conservation. The result-ingpowderwaslyophilizedandstoredat − 20 °Cuntilanalysis.Proteins were extracted from the berry skin powder (0.3 g)following Saravanan and Rose's phenol based protocol [32],whilefor the berry pulp powder(2 g) Sarry'sprotocol wasused[33]. Final pellets were re-suspended in IEF rehydrationsolution (urea 7 M, thiourea 2 M, 4% CHAPS, DTT 130 mM,0.2% IPG buffer 4 – 7, 0.5% IPG buffer 3 – 10). Total proteinconcentration was assessed using Plus One 2DQuant kit (GELife Sciences).IEF was carried out with 60  μ g of proteins per strip using 7 cm long ReadyStrips IPG strips, pH 3 – 10 Non Linear (Biorad)in an Agilent 3100 OFFGEL Fractionator up to 13 kVh, after passive rehydratation for 12 h. Running conditions per stripwere: max voltage 5 kV; max current 50  μ A. Strips were thenequilibrated under gentle agitation, on a stirrer for 15 min,twice, in standard equilibration buffer containing 2% DTT thefirst time, and 2.5% iodoacetamide the second time.SDS-PAGE [34]wasperformed on8 – 16% gradientpolyacryl-amide mini gels using a Mini PROTEAN Tetra cell apparatus(Biorad) at RT. Running conditions per gel were: 10 mA for 10 min,15 mAfor10 min,20 mAuntilbottomofthegel.LMW-SDS marker kit (GE Bioscience) was used as molecular massstandard. Gels were stained with colloidal CBB G-250 [35]. Gelimages were acquired using a ProXpress 2D (PerkinElmer)cooled CCD camera equipped with ProScan software package.The acquisition was performed by bottom illumination withthe use of one UV-to-visible light converter plate. Flat fieldcorrection was used to minimize variance in gel acquisition.The chosen emission filter was ND/2, with exposure time of 2 s, 33  μ m resolution and 16-bits.The PDQuest software package (Biorad) was used for imageanalysis. Three replicate gels were run from each sample. Gelimages were filtered, and spots were detected using the spotdetection tools. The first matching among gels was runautomatically, and then about 20 landmark spots were addedto refine the match, which was finally checked manually andeventually corrected. The normalized volume of each spot wascalculated dividing spot volume value by the sum of totalspot volume values. Total spot volume was calculated by thesoftware,andthisreferredtothesumvolumeofallthespotsonthe gel. Statistical analysis on the resulting normalized spotvalues was performed as described subsequently. 2.8. Mass spectrometry analysis Significantly different spots were excised from the gel andprepared for mass spectrometry as described in [36]. Spectra of proteindigestswereobtainedusingaBrukerUltraflexIIMALDI-TOF/TOFmassspectrometer(BrukerDaltonics,Billerica,USA)inapositivereflectonmodeintherange680 – 4200 Da.Thespectrawere analyzed using the Flex-Analysis 3.3 software package 308  J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 1 ) 3 0 6  –  3 1 5  (Bruker Daltonics) and calibrated internally with the autopro-teolysis peptides of trypsin. Before database search wasperformed, the spectra were depleted of contaminating peaksderiving from both trypsin autodigestion and the digestion of ablank piece of the gel.The MS-Fit ( software packagewasusedtointerprettheMSspectra,throughthePMFmethod[37]. Data were searched against the nrNCBI2010.09.24 data-base (, including 11,894,394 entries, on  Vitis vinifera ,  Saccharomyces cerevisiae , atype of yeast which could be normally found in vineyard, andon GLRaV-1, GVA and RSPaV viruses. The parameters used for the search were: S-carbamidomethyl derivate on cysteine, asfixed modification, oxidation on methionine as variablemodification, and up to two missed cleavage sites for trypsindigestion. The peptide mass tolerance was 20 ppm.After identification, theoretical molecular mass, pI of pro-teins and GO terms were calculated by processing sequenceentries at and pro-teins were assigned to a functional category (FunCat) by theMunichInformationCenterforProteinSequences(MIPS)( accordingtotheirroledescribedinthe literature. 2.9. Statistical analyses 2.9.1. Agronomical performance, berry composition, andinstrumental texture analysis parameters Data obtained by measuring parameters were analyzed byt-test of infected versus uninfected samples. Data expressedas percentage (P%) were subjected to angular transformation[arcsin ( √  P%)] before t-testing. Sprouting index, shoot fertilityand number of clusters per vine were compared by the Mann – Whitney non-parametric U test. 2.9.2. 2D electrophoresis The normalized spot intensity data were exported from thesoftware of srcin and analyzed using SAS statistic software.Normalized spot volumes were analyzed by ANOVA, andsignificantly different spots (p ≤ 0.05) were selected if detect-able in at least two out of three replicates, having a minimumvolume of at least 500 ppm, and showing at least a three-foldvariation. 3. Results 3.1. Plant health status and virus quantification RT-PCR results confirmed the presence of GLRaV-1, GVA andRSPaV in the infected plants, and the healthy status of controlplants. Mean loads of GLRaV-1 and GVA in the infected plantswere 2.5 (SD=0.8) and 0.4 (SD=0.2) viral genomes/100 GAPDHtranscript copies, respectively. The distribution of the viralloads was uniform among the infected plants (SupplementalTable S1). 3.2. Vine agronomical performances and berry chemicalcomposition The infected plants showed a significant decrease in the budburstindexandlower(thoughnotsignificantlyso)shootfertilityand total yield, compared to healthy controls. The berry weightof infected vines was significantly higher than that of healthyplants,duetotheslightlysmallerbunches(Table1).The healthstatusdidnotaffectberrycompositionintermsofsolublesolidsandphenolcontents;however,titratableacidityandresveratrolwere higher in the infected vines (Table 1). 3.3. Instrumental texture analysis The mechanical properties of berry skin and whole berries arereported in Table 2. No significant difference was noticedbetween the mechanical characteristics of infected andhealthy grape skin, due to high data dispersion. Significantdifferences were detected in whole berry cohesiveness andresilience, which are useful parameters to evaluate pulptexture characteristics [38]. Cohesiveness is a measure of thestrength of internal bonds making up the berry body, whileresilience indicates how well the berry regains its srcinalposition after deformation. Table 1  –  Plant agronomical and juice qualitative parameters of healthy and virus infected Nebbiolo vines. Healthy Infected Sample sizes (H, I) or DF Significance Bud burst index 4.5 a ±0.5 3.9±0.3 20,20 ** b No clusters/vine 8.8±2.3 8.0±3.4 20,20 nsShoot fertility (no inflor./shoot) 1.4±0.4 1.2±0.4 20,20 nsBerry weight (g) 1.95±0.09 2.05±0.10 38 **Cluster weight (g) 352±66 339±48 38 nsYield (kg/vine) 3.1±1.0 2.6±1.0 38 nsSoluble solids (°Brix) 23.8±0.6 23.8±0.7 38 nspH 3.02±0.04 3.03±0.04 38 nsTitratable acidity (g/L) 7.6±0.6 8.2±0.5 38 **Total anthocyanins (mg/kg grape) 556±73 551±40 4 nsTotal flavonoids (mg/kg grape) 2687±275 2348±314 4 nsResveratrol (mg/kg grape) 0.25±0.10 0.52±0.10 4 * a Values are means±SD. b ns= P >0.05; *= P ≤ 0.05; **= P ≤ 0.01. 309  J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 1 ) 3 0 6  –  3 1 5  3.4. Protein expression and identification The protein yield per gram of lyophilized tissue powder was2.5 mg for the healthy skin and 2.4 for the infected skin, and0.1 mgforbothpulpsamples.Thedissectionoftheberriesintoskin and pulp, and their subsequent separate proteomicanalysis, allowed for the visualization of a mean of 460 spotin the skin and 380 in the pulp. No significant difference wasdetectedfor proteinyield andtotal spotsbetweenhealthy andinfected status.ANOVAanalysison2Dgelsrevealedsignificantdifferencesbetween healthy and virus-infected grape berries for 7 skinand 11 pulp spots (Fig. 1). Only in the pulp was completedisappearance of 3 out of 11 spots observed. Among theselected spots,numbers4811 and5708 could not be identified.The characteristics of the spots selected as differentiallyexpressed, and the list of proteins identified, are reported inTable 3. The mass of most identified proteins fell within a 25%tolerance limit of published masses. The only exception wasthe C term fragment of cytochrome P450 protein (spot 8102).RSPaV coat protein was identified, together with  V. vinifera proteins, in pulp spots 1604 and 8309.Data regarding the PMF identifications of each spot arereported in Supplemental Table S2. 4. Discussion Grape berry proteomic analysis of healthy and infected plantswas integrated with agronomical analyses to provide acomplete picture of the effects of viral infection on both thefield behaviour and berry quality of Nebbiolo grapevines, animportant and characteristic Italian cultivar. Differences Table 2  –  Skin and whole berry mechanical properties of healthy and virus infected Nebbiolo vines. Parameter Healthy Infected Significance Skin Break force (N) 0.52±0.13 a 0.55±0.13 ns b Break energy (mJ) 0.36±0.15 0.40±0.14 nsYoung's modulus(N/mm)0.35±0.05 0.36±0.06 nsThickness ( μ m) 203±25 200±29 nsBerry Hardness (N) 4.91±0.77 4.96±0.98 nsCohesiveness ( − ) 0.71±0.03 0.73±0.04 *Gumminess (N) 3.45±0.45 3.57±0.56 nsSpringiness (mm) 2.20±0.17 2.25±0.13 nsChewiness (mJ) 7.62±1.36 8.06±1.67 nsResilience ( − ) 0.37±0.02 0.39±0.03 * a Values are means±SD, n=30. b ns= P >0.05; *= P ≤ 0.05. Fig. 1  –  2D gels from  V. vinifera  cv Nebbiolo: healthy (A) and infected (B) berry skins and healthy (C) and infected (D) berry pulps. 310  J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 1 ) 3 0 6  –  3 1 5
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